Smoking remains the number one avoidable cause of death in the United States. Current therapies including nicotine replacement, inhibition of the dopamine system with bupropion, and partial activation of the 4 2 neuronal nicotinic acetylcholine receptor (nAChR) with varenicline have not been particularly effective, with relapse rates at nearly 80% regardless of the treatment drugs or therapies. Furthermore, varenicline, the most efficacious drug, has serious psychiatric side effects. Clearly new treatments are needed. The nAChR most closely associated with the addictive nature of cigarettes is the 4 2 nAChR. Activation of this receptor is the primary mediator of nicotine reward and the selective but low affinity 4 2 nAChR antagonist DH E has been demonstrated to block nicotine self-administration in rodent models. Currently there are no high affinity and selective 4 2 antagonists available to examine as smoking cessation medications. Starting from a small molecule combinatorial library containing over 5 million individual compounds, we have identified selective 4 2 nAChR compounds. Using these screening hits along with medicinal chemistry and molecular modeling, we have identified two lead compounds (2187.397 and 2187.202), one with very high affinity (37 nM) and moderate selectivity (23 fold) in binding assays, and a second with excellent selectivity (>500 fold) but slightly lower affinity (114nM) at the 4 2 nAChR. Both of these compounds are antagonists at 4 2 nAChRs with remarkable in vitro selectivity. In vitro functional assays showed that 202 has 400 fold selectivity at 4 2 over 3 4 nAChR. Furthermore, 202 attenuates nicotine self-administration and blocks nicotine prime-induced reinstatement in rats. Both in vitro selectivity and lack of agonist activity differentiateour compounds from varenicline and might produce fewer side effects. In this Phase I SBIR, we propose to optimize our lead compounds to further improve affinity, selectivity, and drug-like properties. In Specific Aim 1, we will synthesize additional novel analogs based upon the current lead scaffolds. In Specific Aim 2A we will determine binding affinities at 4 2 and 3 4 nAChR, using [3H]epibatidine to bind to membranes produced from HEK cells transfected with the appropriate receptors. Selectivity at additional nAChRs will be determined for the compounds exhibiting the highest affinity for 4 2 and the greatest selectivity against 3 4 nAChR. In Aim 2B we will determine the functional selectivity of compounds showing high affinity and selectivity in the binding assays. Antagonist activity and functional selectivity will be determined by measuring ligand-induced membrane potential changes using a FlexStation on intact HEK cells. Finally in Aim 2C we will measure bioavailability and blood brain barrier penetration to optimize drug-like properties of our lead compounds. These studies will be in preparation for subsequent in vivo efficacy determinations in nicotine reward models as well as additional pharmacokinetic studies, to be completed in a subsequent Phase II application.